# FINAL REPORT

Study Title


## EVALUATION OF THE AB ILITY OF EXP1313100 TO INDUCE CHROMOSOME ABERRATIO NS IN CULTURED PERIP HERAL

HUMAN LYMPHOCYTES

Author

Test Facility WIL Research Europe B.V. Hambakenwetering 7 5231 DD ‘s

- Hert ogenbosch The Netherlands

Laboratory Project Identification Project 505690 Substance 205622/A

- Page 1 of 29

-EXP1313100 Project 505690

- Page 2 - 1. CONTENTS
1. CONTENTS

...................................................................................................................................... 2 2. STATEMENT OF GLP COMPLIANCE

............................................................................................. 4 3. QUALITY ASSURANCE STATEMENT

............................................................................................ 5 4. SUMMARY

........................................................................................................................................ 6 5. INTRODUCTION

.............................................................................................................................. 7 5.1. Preface

...................................................................................................................................... 7 5.2

. Aims of the study

................................................................ ....................................................... 7 5.3. Guidelines

................................................................................................................................ . 8 5.4. Storage and retention of records and materials

................................................................ ........ 8 6. MATERIALS AND METHODS

.......................................................................................................... 8 6.1. Test substance

.......................................................................................................................... 8 6.1.1. Test substance information

............................................................................................. 8 6.1.2. Study specific test substance information

................................................................ ...... 8 6.1.3. Test substance preparation

............................................................................................ 8 6.2. Reference substances

................................................................ .............................................. 9 6.2.1. Negative control

................................................................ .............................................. 9 6.2.2. Positive controls

................................................................ .............................................. 9 6.3. Test system

............................................................................................................................... 9 6.4. Cell culture

................................................................................................................................ 9 6.5. Metabolic activation system

.................................................................................................... 10 6.5.1. Preparation of S9-mix

................................................................................................... 10 6.6. Study design

........................................................................................................................... 10 6.6.1. Dose range finding test

................................................................................................ . 10 6.6.2. Cytogenetic assay

................................................................ ......................................... 11 6.6.3. Chromosome preparation

............................................................................................. 11 6.6.4. Preparation of slides

..................................................................................................... 11 6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay

................................ 12 6.6.6. Analysis of slides for chromosome aberrations

............................................................ 12 6.7. Electronic data capture

................................................................ ........................................... 12 6.8. Interpretation

........................................................................................................................... 12 6.8.1. Acceptability of the assay

............................................................................................. 12 6.8.2. Data evaluation and statistical procedures

................................................................ ... 12 6.9. List of deviations

................................................................ ..................................................... 13 6.9.1. List of protocol deviations

............................................................................................. 13 6.9.2. List of standard operating procedures deviations

......................................................... 13 7. RESULTS

....................................................................................................................................... 13 7.1. Dose range finding test

................................................................ ........................................... 13 7.2. First cytogenetic assay

................................................................ ............................................ 13 7.3. Second cytogenetic assay

...................................................................................................... 14 7.4. Evaluation of the results

................................................................ .......................................... 14 8. CONCLUSION

................................................................................................................................ 15 9. REFERENCES

............................................................................................................................... 15EXP1313100 Project 505690

- Page 3 - TABLES Table 1 Mitotic index of human lymphocyte cultures treated with EXP1313100 in the dose

range finding test

................................................................ .......................................... 16 Table 2 Mitotic index of human lymphocyte cultures treated with EXP1313100 in the first

cytogenetic assay

................................................................ ......................................... 18 Table 3 Chromosome aberrations in human lymphocyte cultures treated with

EXP1313100 in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

................................................................................. 19 Table 4 Chromos ome aberrations in human lymphocyte cultures treated with

EXP1313100 in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

................................................................................. 20 Table 5 Mitoti c index of human lymphocyte cultures treated with EXP1313100 in the

second cytogenetic assay

............................................................................................. 21 Ta ble 6 Chromosome aberrations in human lymphocyte cultures treated with

EXP1313100 in the absence of S9-mix in the second cytogenetic assay (24 h exposure time, 24 h fixation time)

................................................................................. 22 Table 7 Chromos ome aberrations in human lymphocyte cultures treated with

EXP1313100 in the absence of S9-mix in the second cytogenetic assay (48 h exposure time, 48 h fixation time)

................................................................................. 23
APPENDICE S APPENDIX 1 TABLES

........................................................................................................................ 16 APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE

PORTRAITS

................................................................ ................................................. 24 APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

............................................. 26 APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

.............................................................................................. 27 APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

........................................ 28 APPENDIX 6 CERTIFICATE OF ANALYSIS

...................................................................................... 29EXP1313100 Project 505690

- Page 6 - 4. SUMMARY Evaluation of the ability of EXP1313100 to induce chromosome aberrations in cultured peripheral human lymphocytes (with follow up experiment). This report describes the effect of EXP1313100 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of EXP1313100 was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch E00873- 025

- 001 of EXP1313100 was a brown solid. EXP1313100 was soluble in dimethyl sulfoxide. In the first cytogenetic assay, EXP1313100 was tested up to 512 μ g/ mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. EXP1313100 precipitated in the culture medium at this dose level. In the second cytogenetic assay, EXP1313100 was tested up to 125 μ g/ mL for a 24 h continuous exposure time with a 24 h fixation time and up to 100 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. EXP1313100 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of EXP1313100 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that EXP1313100 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Finally, it is concluded that this test is valid and that EXP1313100 is not clastogenic in human lymphocytes under the experimental conditions described in this report.